Abstract：Abstract: Gene therapy is based on the principle of the genetic manipulation of DNA or RNA for treating and preventing human diseases. At present, several gene therapy drugs have entered the stage of clinical research or have come into the market. The advent of gene therapy offers the potential for curing cancer, rare diseases, neurodevelopmental disorders and many other diseases. Safe and effective gene delivery is a pre-requisite for successful gene therapy. Adeno-associated virus (AAV) vector is one of the most popular vectors for human gene therapy. It’s the only viral vector that can integrate into the specific site of human genome. The adeno-associated virus site 1 (AAVS1) has been proven to be a safe harbor of gene integration. Herein, the transgene was site-specifically integrated into the AAVS1 site of human T lymphocyte genome with the Rep protein as the recombinase, and the AAVS1-integration rate of mixed cells was quantified by standard curve quantitative method. Two rounds of PCR were carried out. First the sequence containing the ITR-AAVS1 binding site was amplified by conventional PCR. Then the first-round PCR products were used as templates in the second round quantitative PCR. Through this method, AAVS1 site integration efficiency can be quickly and quantitatively analyzed.
引用本文:
张轶岭, 王飞飞, 张 春, 李文生. 人细胞基因组AAVS1位点基因定点整合率的荧光定量PCR检测方法[J]. 生命科学研究, 2018, 22(6): 431-437.
ZHANG Yi-ling, WANG Fei-fei, ZHANG Chun, LI Wen-sheng. Quantitative Fluorescence PCR Method for AAVS1 Site-specific Integration Rate Analysis. Life Science Research, 2018, 22(6): 431-437.
